Naphthalene-based RNA Editing Inhibitor Blocks RNA Editing Activities and Editosome Assembly in Trypanosoma brucei

Abstract

RNA editing, catalyzed by the multiprotein editosome complex, is an essential step for the expression of most mitochondrial genes in trypanosomatid pathogens. It has been shown previously that Trypanosoma brucei RNA editing ligase 1 (TbREL1), a core catalytic component of the editosome, is essential in the mammalian life stage of these parasitic pathogens. Because of the availability of its crystal structure and absence from human, the adenylylation domain of TbREL1 has recently become the focus of several studies for designing inhibitors that target its adenylylation pocket. Here, we have studied new and existing inhibitors of TbREL1 to better understand their mechanism of action. We found that these compounds are moderate to weak inhibitors of adenylylation of TbREL1 and in fact enhance adenylylation at higher concentrations of protein. Nevertheless, they can efficiently block deadenylylation of TbREL1 in the editosome and, consequently, result in inhibition of the ligation step of RNA editing. Further experiments directly showed that the studied compounds inhibit the interaction of the editosome with substrate RNA. This was supported by the observation that not only the ligation activity of TbREL1 but also the activities of other editosome proteins such as endoribonuclease, terminal RNA uridylyltransferase, and uridylate-specific exoribonuclease, all of which require the interaction of the editosome with the substrate RNA, are efficiently inhibited by these compounds. In addition, we found that these compounds can interfere with the integrity and/or assembly of the editosome complex, opening the exciting possibility of using them to study the mechanism of assembly of the editosome components.Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major are three major trypanosomatid pathogens that cause hundreds of thousands of deaths and infect millions of people in tropical and subtropical areas of the world (1). Current trypanocidal drugs have a number of limitations such as high rate of toxicity, low rate of efficacy, and drug resistance (2, 3). Therefore, it is important to look for a drug that is effective and does not produce harmful side effects. RNA editing is a unique posttranscriptional modification of mitochondrial mRNAs that is shared in all trypanosomatid pathogens (4, 5). Modification of specific editing sites, dictated by complementary guide RNAs (gRNAs), 5 constitutes essential steps to ensure the production of translatable mRNAs that encode essential components of the mitochondrial respiratory system. Although gRNAs specify the number of uridylates (Us) to be added or deleted by base pairing at each editing block (6, 7), a 1.6-MDa multiprotein complex, the editosome, is responsible for catalysis of different steps of RNA editing. Although the complete composition of the editosome is being elucidated, most purified functional editosomes contain over 20 proteins (8,9). The editosomes differ in their compositions, having at least three different complexes that sediment at ϳ20 S on glycerol gradie...

Functional characterization of two paralogs that are novel RNA binding proteins influencing mitochondrial transcripts of Trypanosoma brucei

“…This complex encompasses the cascade of enzymatic activities for U-insertion/deletion events: An endonuclease cleaves the mRNA at the editing site dictated by the gRNA, which also provides information to RECC for how many U's are inserted or deleted here by a terminal uridylyltransferase (TUTase) or exonuclease, respectively; in the last step, the processed site is sealed within the mRNA by an RNA ligase. These activities are being explored as potential drug targets for treating these pathogens in their mammalian hosts (Durrant et al 2010;Moshiri et al 2011).…”

The crystal structure of an oligo(U):pre-mRNA duplex from a trypanosome RNA editing substrate

“…Safer and more effective drugs are needed. These parasites share a RNA editing pathway (Benne et al 1986) that is absent in humans, essential for parasite survival (Schnaufer et al 2005), and a potential drug target (Amaro et al 2008;Liang and Connell 2010;Moshiri et al 2011). Large (z20S) ribonucleoprotein editing complexes (Simpson et al 2004;Carnes et al 2008)-similar in dimensions and complexity to the ribosome and spliceosome (Golas et al 2009;Li et al 2009)-insert or delete uridylates (Us) through enzyme cascades (Seiwert and Stuart 1994;Adler and Hajduk 1997) that are under the direction of hundreds of different guide RNAs (gRNAs) Pollard et al 1990;Ochsenreiter et al 2007) that bind antiparallel to the pre-mRNA at specific sites.…”

Pilot-Scale Compound Screening against RNA Editing Identifies Trypanocidal Agents

“…18,20 The purified editosome was then analyzed and quantified via Western blotting. The most active editosome fractions were identified using the fluorescence resonance energy transfer (FRET)-based in vitro editing assay.…”

“…[15][16][17][18] However, because of the high editosome concentrations required for completion of an RNA editing cycle in vitro, two of the potent inhibitors were found to affect RNAprotein interactions instead of inhibiting the ligase itself. 18 Another study identified five inhibitors of insertion RNA editing, with two specifically acting at or prior to the endonuclease step. 19 In the present study, we used our highthroughput screening (HTS) assay 17 that targets RNA editing to screen a library of pharmacologically active compounds from Sigma (LOPAC 1280 ; Sigma, St. Louis, MO).…”